Construction Of STM Mutants And Structure And Functional Study Of LuxS Gene Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumonia,causing severe economic losses globally in the swine industry.A. pleuropneumoniae can be divided into 15 serotypes based on the differences of capsular polysaccharide and lipopolysaccharide and the virulence and distributions of serotypes are various.Main virulence factors of A.pleuropneumoniae include capsular polysaccharide,lipopolysaccharide,outer membrane proteins,transferrin binding proteins, RTX toxins,adhesins,urease and proteases.To investigate the pathogenesis of A. pleuropneumoniae,the characterization of genes involved in the infection of this bacteria is needed.This study focus on construction of signature-tagged mutagenesis(STM) mutants and the structure and functional role of luxS gene of A.pleuropneumoniae.1.Construction of STM mutants of A.pleuropneumoniaeSTM is a powerful approach to identify genes associated with virulence of the pathogenic bacteria through negative selection.This approach has been successfully used in more than 30 pathogenic bacteria.Many genes involved in A.pleuropneumoniae infection have been screened out by STM study,and nalidixic acid resistant A.pleuropneumoniae strains have been obtained in our laboratory.In this study,by using a sophisticated biparental mating method and antibiotic selection and PCR selection, 215 mutants containing four of the 48 STM tags(Tag 5,Tag 6,Tag 9,and Tag 10) were obtained,within which there are 49 mutants containing Tag 5,55 mutants containing Tag 6,59 mutants containing Tag 9 and 52 mutants containing Tag 10.Meanwhile,the process of mating and successful rate were summarized.This will contribute to STM study and identification of new functional genes from A.pleuropneumoniae.2.The structure and functional study of iuxS of A.pleuropneumoniaeQuorum sensing is known to be a mechanism by which bacteria regulate their behaviors through certain signals secreted by themselves or their hosts.LuxS is known to be involved in the interspecies quorum sensing by mediating the production of AI-2 signal molecule and play important roles in regulating various behaviors of bacteria.In this study,A.pleuropneumoniae was tested to be able to produce AI-2 like molecule.A luxS deletion mutant strain was constructed by homologous replacement,and its growth features,biofilm formation and virulence were characterized.The luxS mutant showed obvious growth deficiency when cultured in the serum-free medium.Biofilm formation of the mutant strain was significantly enhanced.In a mouse infection model,the 50%lethal dose of the mutant strain was increased up to 96 folds,and the ability to colonize in different mouse tissues was significantly decreased.Down-regulation of apxâ…¡A,a major virulence factor of A.pleuropneumoniae,may contribute to the virulence attenuation of the mutant strain.Our data demonstrated that LuxS is functional in the AI-2-mediated quorum sensing in A.pleuropneumoniae and LuxS may regulates different behaviors including biofilm formation and virulence of this important pathogen.